Circulating Re-Entrant Waves Promote Maturation of Hipsc-Derived Cardiomyocytes in Self-Organized Tissue Ring

ARTICLE https://doi.org/10.1038/s42003-020-0853-0 OPEN Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring Junjun Li 1,11, Lu Zhang2,11, Leqian Yu3, Itsunari Minami4, Shigeru Miyagawa1, Marcel Hörning 3,10, Ji Dong5, 1234567890():,; Jing Qiao3, Xiang Qu1, Ying Hua1, Nanae Fujimoto1, Yuji Shiba6, Yang Zhao7, Fuchou Tang5, Yong Chen3,8, ✉ ✉ ✉ Yoshiki Sawa1 , Chao Tang 2 & Li Liu1,9 Directed differentiation methods allow acquisition of high-purity cardiomyocytes differ- entiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC- derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen- consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs. 1 Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. 2 Center for Quantitative Biology and Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, 100871 Beijing, China. 3 Institutes for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan. 4 Department of Cell Design for Tissue Construction Faculty of Medicine, Osaka University, Osaka 565-0871, Japan. 5 Biomedical Pioneering Innovation Center, College of Life Sciences, Peking University, 100871 Beijing, China. 6 Department of Regenerative Science and Medicine, Institute for Biomedical Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-0821, Japan. 7 State Key Laboratory of Natural and Biomimetic Drugs, The MOE Key Laboratory of Cell Proliferation and Differentiation, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, 100871 Beijing, China. 8 PASTEUR, Département de chimie, école normale supérieure, PSL Research University, Sorbonne Universités, UPMC Université Paris 06, CNRS, Paris 75005, France. 9 Department of Drug Discovery Cardiovascular Regeneration, Osaka University Graduate School of Medicine, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. 10Present address: Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, 70569 Stuttgart, Germany. 11These authors ✉ contributed equally: Junjun Li, Lu Zhang. email: [email protected]; [email protected]; [email protected] COMMUNICATIONS BIOLOGY | (2020) 3:122 | https://doi.org/10.1038/s42003-020-0853-0 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-0853-0 lthough human-induced pluripotent stem cell (hiPSC) cardiomyocytes aggregated and formed a thick tissue ring within Aderived cardiomyocytes have been proposed as an abun- 2 days (432.72 ± 56.18 µm on day 2, Fig. 1a–c and Supplementary dant resource for tissue engineering, drug screening, and Fig. 1). As indicated by the genetically encoded calcium indicator regenerative-medicine applications1, they exhibit characteristics GCaMP3, we found looped activation propagations within the different from adult human cardiomyocytes, including immature SOTRs (i.e., ReWs; Supplementary Movie 1), with zero to two sarcomere structure and morphology, a fetus-like gene expression ReWs present in one SOTR (Fig. 1d, e and Supplementary profile, and inadequate Ca2+-handling properties2–4. The Movie 2). Besides circular geometry, other geometries have also immature nature of hiPSC-derived cardiomyocytes potentially been tested, such as 4-point star, 5-point star (Supplementary hinders their reflection of adult heart physiology for disease Fig. 2a), and the geometry between rectangular and circular could modeling and drug assessment. also be obtained. However, the formation of the tissues To achieve higher degrees of hiPSC-derived cardiomyocytes throughout different areas of these templates was less homo- maturation and function, a variety of methods have been devel- geneous than that of the tissues in the circular geometry. In oped, including dynamic culture5, three-dimensional (3D) engi- addition, the circular ring tissues were more equally and stably neered heart tissue6–8, 3D printing9, addition of factors10, organized while maintaining the ReWs with a higher ratio extracellular matrix10,11, and external stimulation. Electrical sti- (Supplementary Fig. 2b). Thus, we chose the circular ring to mulation, especially high-frequency pacing, has long been sug- engineer cardiac tissues. Fluorescence-activated cell sorting gested as an effective method for maturing muscle cells3,12–15, (FACS) (Supplementary Fig. 1b), voltage dye staining, and which upregulates cardiac markers13,14,16, enhances Ca2+-hand- immunostaining data indicated that the SOTRs were mostly ling properties3, and promotes cardiomyocyte alignment17. ventricular cardiomyocytes and few fibroblast cells (Supplemen- During this process, appropriate stimulation protocols are tary Figs. 3 and 4). required to minimize possible tissue damage18. Additionally, it A trace recording (Fig. 1d, e) indicated no long rest periods remains challenging to rapidly pace cardiomyocyte tissues (≥2 between successive ReWs, resulting in higher beating rates in Hz) over long time19 due to possible side effects, such as heavy SOTRs capable of sustaining a higher number of ReWs (Fig. 1e, metal poisoning, electrolysis, pH shift, and the generation of f). The spontaneous beating rate of SOTRs with zero ReW was reactive oxygen species (ROS)20,21. Moreover, upscaling for mass 0.21 ± 0.10 Hz on day 6 and remained stable for 2 weeks, whereas stimulation is either difficult or requires high level of power the rates of SOTRs with one or two ReWs on day 6 were much consumption19. As an important complement, mechanical sti- higher (2.40 ± 0.49 and 3.30 ± 0.39 Hz, respectively). Notably, the mulation can promote cardiomyocyte maturation, with cyclic wave speed in the ReW groups was much lower (~2 cm s−1) mechanical stress applied by external stretching devices capable than that of the spontaneous beating group (0 ReW; 5.93 ± of forcing muscle-cell assembly into structurally and functionally 1.50 cm s−1). It is possible that the slower speed together with the aligned 3D syncytium22, thereby affecting their gene expres- shortened pacing interval and refractory period might have been sion23–25 and Ca2+ cycling26. caused by the higher beating frequency in the ReW groups On the other hand, spiral waves [re-entrant waves (ReWs)] relative to that in the zero ReW group (Supplementary Fig. 5 and within cardiac tissue have long been investigated as a model for Supplementary Movie 3), which agrees with previous reports arrhythmia studies27–31, and the rapid beating caused by in vivo associated with excitable media32–36. Additionally, after the ReW spiral wave could lead to dysfunction of heart and even death of a was stopped, the speed of spontaneous beating in the two ReW patient. However, when used in an in vitro condition, the rapid group increased significantly (p = 0.01) to more than 10 cm s−1 beating caused by spiral wave, similar to rapid electrical stimu- (Fig. 1g). lation14, could be beneficial for cardiomyocytes’ maturation. ThebeatingratesoftheoneandtwoReWgroupsincreased In this study, we create a platform capable of promoting rapid slightly over 2 weeks of culturing, reaching 3.03 ± 0.68 and 3.92 formation of hiPSC-derived cardiomyocytes into 3D self- ± 0.69 Hz on day 14, respectively. After a 6-day culture period, organized tissue rings (SOTRs), where propagation of an action 83.8% of 204 SOTRs had one or two ReWs, 14.2% had zero potential in the form of ReWs can spontaneously originate and ReW, and 2% had three ReWs; however, these percentages travel around the closed-loop circuit, thereby making the cardi- changed after 2 weeks of culture to 28.5% with zero ReW, 48.9% omyocytes beat at a high frequency (~2–4 Hz) continuously and with one ReW, 22.6% with two ReWs, and no SOTRs with three robustly up to more than 89 days without any external stimula- ReWs (Fig. 1h). Noise and disturbances, such as those during tion. Additionally, we found that the beating frequency and the medium changing, might have provoked changes in and/or ReW speed can be adjusted by changing the diameter of the ring. disappearance of ReWs. In the long-term culture, we found that Furthermore, we construct a mathematical model in order to ReWs could be maintained in SOTRs for >89 days (Supple- elucidate the origination, propagation, and long-term behavior of mentary Fig. 6). In addition to the static culture, dynamic the ReWs, with the model ultimately agreeing well with the culture of SOTRs on a rotary shaker has been applied to check experimental data. After 2 weeks of culture, the SOTRs with the effect on ReWs. During the culture, we did find the ReWs demonstrate

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